Retroviral full-length RNA serves two important roles in viral replication: the template for Gag/Gag-Pol translation and the genome in the virion. Our previous results showed that most HIV-1 particles have two copies of viral RNA genome, indicating that the packaging is tightly regulated. To examine whether HIV-1 RNA packaging is regulated by RNA mass, we tested viral genomes of different lengths and determined that HIV-1 packaged two copies of RNA regardless of whether the RNAs were 3 kb or 17 kb. To test whether HIV-1 recognizes two copies of RNA or one dimeric RNA, we generated HIV-1 genomes that could form a self-dimer and found that only one copy of the self-dimer RNA was packaged. These results indicated that HIV-1 packaging is not regulated by RNA mass but by recognizing a dimeric RNA. We have now defined the sequences necessary and sufficient for packaging RNA into HIV-1 virion. We are examining whether HIV-1 RNA-packaging specificity can be altered by RNA-binding proteins and use this strategy to study how RNA packaging is regulated. Additionally, we will investigate HIV-1 RNA structures in the virus producer cells, determine the role of several highly structured RNA elements in HIV-1 replication, and delineate the host factors that affect HIV-1 RNA nuclear export and the dynamics of HIV-1 RNA export from the nucleus to the cytoplasm. These experiments seek to gain insights into how HIV-1 RNA serves its functions. __BACKGROUND: The full-length HIV-1 RNA (hereafter referred to as HIV-1 RNA) serves as a template for Gag/Gag-Pol translation and as the virion genome. HIV-1 RNA needs to negotiate through the complex cellular regulation of the host to be exported from the nucleus to the cytoplasm. Once exported, HIV-1 RNA can be translated and/or packaged and needs to strike a balance between these two functions. In this project, we sought to gain a better understanding of how HIV-1 RNA serves its roles. __As an unspliced RNA, HIV-1 RNA needs to bypass the cellular gatekeepers to be exported from the nucleus and reach the cytoplasm. HIV-1 RNA contains an RNA structure, the Rev responsive element (RRE). The viral protein Rev binds to the RRE and interacts with the host protein CRM1 to allow for the export of HIV-1 RNA. Recent studies revealed that the regulation of RNA export may be more complex than previously envisioned and may involve multiple host factors other than CRM1 and RanGTP. We aim to better define the process of HIV-1 RNA export by identifying host factors involved in the nuclear export and by determining the dynamics of the export process. __It has long been proposed that full-length HIV-1 RNA assumes different structures in the cell to be either translated or packaged; however, little is known about cellular HIV-1 RNA structures. We are collaborating with Drs. Kevin Weeks (U. North Carolina) and Robert Gorelick (Leidos), who have probed the HIV-1 RNA structure in virions, to study cellular HIV-1 RNA structures. Portions of the virion HIV-1 RNA are highly structured. Some RNA structures, such as the RRE, have known functions, whereas others do not. We will examine whether five recently identified RNA structures with unknown functions play a role in HIV-1 replication and determine the HIV-1 sequence required for packaging heterologous RNAs into HIV-1 particles. HIV-1 tightly regulates genome packaging to achieve two copies of HIV-1 RNA (one dimer) in most viral particles. To dissect the mechanisms that regulate genome packaging, we tested whether we can replace the function of the NC domain by modifying Gag proteins. These studies are designed to provide insights into the regulation of HIV-1 RNA and to gain a better understanding of viral replication. __Several of the experiments described in this project use a single-virion assay that we previously developed; this assay allows us to examine the HIV-1 RNA genome in individual particles. Briefly, we modified the HIV-1 genome so that only the full-length RNA contained stem-loop sequences, BSL and MSL, which are specifically recognized by the E. coli BglG protein or the bacteriophage MS2 coat protein, respectively. Additionally, some of the HIV-1 genomes encoded untagged Gag whereas others encoded Gag fused to a cerulean fluorescent protein (CFP). We cotransfected HIV-1 genomes encoding untagged Gag and Gag-CFP, and RNA-binding proteins tagged with fluorescent proteins into human 293T cells, harvested viral particles, and examined the viral particles using fluorescent microscopy. HIV-1 particles were identified by the CFP signals and viral RNAs were identified by either mCherry or yellow fluorescent protein (YFP) signals from the RNA-binding proteins. Using this method, we determined that most (90%) of the HIV-1 virions contained viral RNA; furthermore, two copies of the viral genome were packaged in each particle. Thus, HIV-1 RNA packaging is tightly regulated. __ACCOMPLISHMENTS: Our previous results showed that HIV-1 RNA packaging is tightly regulated. To explore the possible mechanisms, we hypothesized that HIV-1 genome packaging could be regulated by the RNA mass or by the RNA copy number. We found that two copies of RNA genomes were packaged into viral particles regardless of whether the HIV-1 RNAs were 3 kb or 17 kb, indicating that HIV-1 RNA packaging is not regulated by the mass of the RNA. Based on these results, we further examined whether HIV-1 genome packaging is regulated by the recognition of two copies of RNA or one dimeric RNA. To distinguish between these two possibilities, we generated HIV-1 genomes that contained a duplicated dimerization signal and thus could form a self-dimer. We found that HIV-1 can package only one copy of RNA if it forms a self-dimer. These results indicated that HIV-1 genome encapsidation is regulated by packaging one RNA dimer. __We also examined the sequence necessary and sufficient for packaging RNA into HIV-1 virions. We performed deletion analyses to define the 5' untranslated region (UTR) and 5' portion of the gag gene that are important for packaging. We then inserted viral sequence into heterologous nonviral RNA and found that RNA encompassing the 5' UTR and 5' gag gene can be packaged efficiently into viral particles. This study defined the sequence required for HIV-1 genome encapsidation. __[Corresponds to Hu Project 1 in the July 2016 site visit report of the HIV Dynamics and Replication Program]